Phosphoric acid, mixed esters with Bu alc. and ethylene glycol

Endpoint summary

Description of key information

Phosphoric acid, mixed esters with butyl alcohol and ethylene glycol was administered daily in graduated doses to 3 groups of test animals for 90 days. Animals of an additional control group were handled identically as the dose groups but received sterile water, the vehicle used in this study. The 4 groups comprised 10 male and 10 female rats.
Based on the results of this study, the no-observed-adverse-effect-level (NOAEL) for the test item, Phosphoric acid, mixed esters with butyl alcohol and ethylene glycol, was considered to be 200 mg/kg body weight/day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 August 2018 to 18 May 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

OECD Guideline No. 408, adopted on 25 June 2018

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

25 June 2018.

Deviations:

no

Principles of method if other than guideline:

Not Applicable

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:Clariant India Ltd and DEH8006819
- Expiration date of the lot/batch:28.10.2019
- Purity test date:01.03.2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:Ambient (21 to 29°C)
- Stability under test conditions: The test item formulations at 0.5 mg/mL and 40 mg/mL were stable up to 6 hours at room temperature
- Solubility and stability of the test substance in the solvent/vehicle:Clearly miscible with Milli-Q water at the concentration 200 mg/mL and stable upto 6 hours in ambient condition.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:Miscible with Millli-Q water to get desired concentration of 5,10 and 20 mg/mL

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Rat is one of the recommended species by regulatory agencies for conducting toxicity assessment studies among rodents.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: In-house bred animals
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 7 weeks
- Weight at study initiation: 120 to 150g for males; 140 to 170g for females
- Housing: Maximum of two animals of same sex
- Diet (e.g. ad libitum):Altromin maintenance diet for rats and mice (manufactured by Altromin Spezialfutter GmbH & Co. KG)
- Water (e.g. ad libitum):Deep bore-well water passed through Reverse Osmosis Unit
- Acclimation period: Six Days

ENVIRONMENTAL CONDITIONS
- Temperature (°C):19.3ºC to 23.1 ºC
- Humidity (%):47% to 67%
- Air changes (per hr):12 to 15 air changes per hour
- Photoperiod (hrs dark / hrs light):12 hours fluorescent light and 12 hours dark cycle

IN-LIFE DATES: From: To: 18 August 2018 to 16 January 2019

Route of administration:

oral: gavage

Details on route of administration:

The test formulations/vehicle was administered through oral gavage using oral intubation cannula attached to disposable syringe

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle: Milli-Q water is one of the commonly used vehicle for toxicity studies
- Concentration in vehicle: Low dose: 5 mg/mL; Mid dose: 10 mg/mL; High dose: 20 mg/mL
- Amount of vehicle : 10 mL/kg body weight
- Purity: 100%

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Formulation analysis for concentration verification was performed for all the dose formulations in 1st month, 2nd month and 3rd month of the study according to the following scheme:
For dose concentration verification analysis, the prepared formulations were sampled in duplicate sets (60 mL × 2 sets) from each dose level during week 1, 2nd months and 3rd months of the treatment period.
The collected samples were transferred to Analytical Chemistry Department of Bioneeds India Private Limited, for dose concentration analysis. One set of aliquot of each formulation was analysed. The second aliquot was stored as a backup purpose at established stability conditions. The second set of samples was discarded as the analysis results of first set of samples were within the limits. Formulations were considered acceptable as the mean results were within the range of 90 to 110% of the nominal concentration and the relative standard deviation (% RSD) is ≤10%.

Duration of treatment / exposure:

90 consecutive days

Frequency of treatment:

once daily

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Vehicle control

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Remarks:

Low Dose

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

Mid

Dose / conc.:

200 mg/kg bw/day (actual dose received)

Remarks:

High

No. of animals per sex per dose:

10 animals/group/sex

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The doses of 50, 100 and 200 mg/kg/day was selected as low (G2), mid (G3) and high dose (G4/G4R) levels for test item Hordaphos MDGB as per recommendation from sponsor
- Rationale for animal assignment (if not random):The animals for the experiment were weighed and arranged in ascending order of their body weights. The body weight stratified rats were distributed to all the experimental groups using Microsoft Excel Spreadsheet, such that body weight variation of animals selected for the experiment did not exceed ±20% of the mean body weight of each sex.
- Rationale for selecting satellite groups: The animals for the experiment were weighed and arranged in ascending order of their body weights. The body weight stratified rats were distributed to all the experimental groups using Microsoft Excel Spreadsheet, such that body weight variation of animals selected for the experiment did not exceed ±20% of the mean body weight of each sex.
- Post-exposure recovery period in satellite groups: 28 Days recovery period without treatment

Positive control:

Not Applicable

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once Daily
- Cage side observations checked in table [No.1] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly once

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly once

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Weekly once

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Once before treatment on the day of grouping and during week 13 for main group animals and week 17 for recovery group anaimals
- Dose groups that were examined: Main group animals (G1 and G4), Recovery group animals (G1R and G4R)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At termination
- Anaesthetic used for blood collection:Mild Isoflurane anaesthesia
- Animals fasted: Yes
- How many animals: All the animals prior to the sacrifice
- Parameters checked in table [No.10] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:At termination
- Animals fasted: Yes
- How many animals:All the animals prior to the sacrifice
- Parameters checked in table [No.11] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine:At termination
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table [No.12] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Week 13 and Week 17
- Dose groups that were examined: Main groups: G1 and G4;
Recovery group: G1R and G4R
- Battery of functions tested: sensory activity, grip strength and motor activity

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 1 in Appendix 19 - Pathology report)

HISTOPATHOLOGY: Yes (see table 2 in Appendix 19 - Pathology report)

Other examinations:

-Vaginal smear was examined from all the females on the day of sacrifice.

-The blood samples for estimation of T3, T4 and TSH were collected at termination.

Statistics:

The data was subjected to statistical analysis using SPSS software version 22. Body weight, percent change in body weight with respect to day 1, feed consumption, absolute organ weights and ratios, hematological and clinical chemistry estimations, FOB (rearing, urination, defecation and excessive grooming) and urine analysis (urobilinogen, pH and specific gravity), parameters were subjected to statistical analysis. One way ANOVA followed by Dunnett’s post test was done for different treatment groups comparing with the control group data. Comparison of means between recovery groups was done using ‘t’ test. All analysis and comparisons were evaluated at the 95% level of confidence (p<0.05).

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs were noted in any of the treated group animals in either sex.

Mortality:

no mortality observed

Description (incidence):

No mortality/morbidity were noted in any of the treated group animals in either sex.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Statistical significant (p<0.05) lower mean body weight was noted in G2 males (Days 50, 57), G4 males (Days 43, 50, 57, 64, 71, 78, 85), G2 females (Days 50, 64, 71, 78, 85, 90), G4 females (Days 50, 57, 64, 71, 78, 85, 90), G4R males (Days 50, 57, 64, 71, 78, 85); lower percent change in body weight with respect to day 1 was noted in G2 males (Days 1-43 to 1-85), G3 males (Days 1-43 to 1-64), G4 males (Days 1-29 to 1-90), G2 females (Days 1-50), G4 females (Days 1-50 to 1-90), G4R males (Days 1-43 to 1-78), G4R females (Days 1-22 to 1-85). The lower mean body weight up to a maximum of 13% in males and 7% in females was noted; this lower changes in the body weights was not associated with concomitant variation in feed consumption. Hence, the noted change in body weight is considered as non-adverse in the absence of any associated clinical signs or changes

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

No treatment related changes in feed consumption were noted when compared to vehicle control group. However, few incidences of statistically significant lower feed consumption was noted in G2 males (weeks 10, 11), G3 and G4 females (week 3) and G4R males (week 3). These isolated occurrences in lower feed consumption is considered incidental due inconsistency and/or due to lack of dose responsiveness

Food efficiency:

not examined

Description (incidence and severity):

Not applicable

Water consumption and compound intake (if drinking water study):

not examined

Description (incidence and severity):

Not applicable

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No ocular changes were observed during ophthalmoscopic examination

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment related changes were observed in haematology parameters when compared to vehicle control group. However, the following statistically significant (p<0.05) variations were noted.
In males, increase in MPV (G4), absolute and percent neurtrophils (G2), APTT (G2) was noted.
In females, increase in RBC (G2), haemoglobin (G2), haemotocrit (G2), MPV (G3, G4), absolute & percent eosinophils (G3), reticulocyte count (G4R), absolute reticulocyte count (G4R); decrease in APTT (G2, G4, G4R) was noted.
The variations noted in main groups are considered incidental due to lack of dose dependency; the variations noted in recovery groups are also considered incidental as the similar variations were not noted in the other sex. Further, the variations noted could be due to random biological variation

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment related changes were observed in clinical chemistry parameters when compared to vehicle control group. However, the following statistically significant (p<0.05) variations were noted.
In males, increase in triglycerides (G4), phosphorous (G4), sodium (G3, G4), glucose (G4R), HDL (G4R), BUN (G4R) and urea (G4R) was noted.
In females, increase in total protein (G4), calcium (G3), cholinesterase (G2, G3), sodium (G3, G4); decrease in ALP (G4), calcium (G4) was noted.
The variations noted in main groups are considered incidental due to lack of dose dependency; the variations noted in recovery groups are also considered incidental as the similar variations were not noted in the other sex. Further, the variations noted could be due to random biological variation in the absence of changes in associated organs/tissues

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant increase in pH (G2, G4 and G4R males); decrease in specific gravity (G3 and G4 females) was noted. The observed variations noted in main groups are considered incidental in the absence of dose responsiveness. The increase in pH noted in G4R males is also considered incidental as the same was not noted at the end of treatment period in main groups and also was not noted in other sex (recovery females)

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No treatment related changes were observed in neurological/functional examination test when compared to vehicle control group

Immunological findings:

not examined

Description (incidence and severity):

Not applicable

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Absolute organ weight: In males, decrease in thymus (G4); increase in thyroid with parathyroid (G2, G3, G4 and G4R) was noted. In females, decrease in spleen (G2), uterus (G2), liver (G2, G4R), lungs (G2); increase in thymus (G4R) was noted.
Relative organ weight: In males, increase in spleen (G2, G4), testes (G4), epididymis (G4), heart (G4), kidney (G4), brain (G2, G4), liver (G4), thyroid & parathyroid (G2, G3, G4 and G4R) was noted. In females, increase in kidneys (G4), thyroid & parathyroid (G4), thymus (G4R) was noted.
All the variations noted are considered incidental and not test item-related as there were no associated histopathological findings in any of the high dose group animals.
Statistically significant decrease in fasting body weight was noted in G4 males, G2 and G4 females. In the absence of dose relationship, the observed variation is not considered to be treatment related

Gross pathological findings:

no effects observed

Description (incidence and severity):

No treatment related gross pathological changes were noted

Neuropathological findings:

not examined

Description (incidence and severity):

Not applicable

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment related histopathological findings were identified in the study.
Lesions considered to be spontaneous and incidental were observed in treated and control rats. These lesions consisted of interstitium mononuclear cells infiltrate in kidneys and prostate gland, golden-brown pigment in red pulp of spleen and neutrophils infiltrate in harderian gland.

The presence of a small amount of intracytoplasmic pigment (golden-brown pigment within the splenic red pulp macrophages is a common background finding in rodents.
Isolated incidence of cystic tubular dilation in kidneys, necrosis of exocrine pancreas and mononuclear cells infiltrate in tongue seen only in high dose group (G4) animals. These lesions were considered to be spontaneous because either it’s unilateral in nature and or lack of consistency.
One male animal from high dose group (G4) showed murine progressive cardiomyopathy.
Brian et al., reported that rodent progressive cardiomyopathy is a spontaneous, chronic and progressive myocardial disease that occurs in many strains of rats and mice with some variability in incidence and severity.
A presence of ectopic thymus and ultimobranchial cyst in thyroid, accessory cortical nodule in adrenal and cyst in pituitary gland were congenital lesions and it does not have toxicological significance.

Histopathological findings: neoplastic:

not examined

Description (incidence and severity):

Not applicable

Other effects:

no effects observed

Description (incidence and severity):

-No changes were observed in the estrus cycle at any of the tested dose group females in main group and recovery group
-No statistically significant variations noted in any of the thyroid hormone assessed (T3, T4 and TSH) when compared with vehicle control group.

Details on results:

Based on the observed results, the No Observed Adverse Effect Level (NOAEL) of test item Hordaphos MDGB is 200 mg/kg/day when administered for a period of 90consecutive days by oral (gavage) route to Sprague Dawley under the test conditions and doses employed

Key result

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

mortality

ophthalmological examination

organ weights and organ / body weight ratios

urinalysis

other: Vaginal smear Examination for females at termination. T3, T4 and TSH estimation for main and recovery group animals

Key result

Critical effects observed:

no

Not applicable

Conclusions:

Based on the observed results, the No Observed Adverse Effect Level (NOAEL) of test item Hordaphos MDGB is 200 mg/kg/day when administered for a period of 90 consecutive days by oral (gavage) route to Sprague Dawley under the test conditions and doses employed.

Executive summary:

The objective of this study was to assess the toxic potential of the test item Hordaphos MDGB when administered for a minimum period of 90 consecutive days repeatedly by oral (gavage) to Sprague Dawley rats. This study provides information on major toxic effects, target organs, possibility of cumulative effects and also the reversibility of effects after 28 days recovery period and an estimate of the No Observed Adverse Effect Level (NOAEL).

A total of 120 Sprague Dawley rats (60males +60females) were distributed to 4 main and 2 recovery groups. Each main groupand recovery groupconsisted of 10 animals/sex/group.The animals allocated togroupsG2, G3 and G4/G4R were administered with oral doses of 50, 100 and 200 mg/kg body weight/day of test item, respectively through oral gavage.The animals allocated to group G1/G1Rreceived vehicle sterile water (Milli-Q water). The vehicle/test item formulations were administered at an equivolume of 10 mL/kg.

Formulation analysis for concentration verification was performed for all the dose formulations in 1^stmonth, 2^ndmonth and 3^rdmonth of the treatment period. All formulations were within±10% of the target concentrations and the relative standard deviation (RSD) was less than 10%.

All animals were observed once daily for clinical signs and twice daily for mortality; weekly for detailed clinical examination, body weight and feed consumption; ophthalmoscopic examination was performed during acclimatization and during week13for main groups (G1andG4) and during week 17 for recovery groups (G1R and G4R). Neurological/Functional observational battery was performed during week13 for control and high dosemain group animals(G1 and G4) and during week17 for recovery group animals(G1R and G4R).

At the end of treatment and recovery period (day 91 for main group and day 119 for recovery group), blood and urine samples were collected and analyzed. Subsequently, the animals were sacrificed and subjected to gross pathological examination and the organs were collected, weighed and preserved. The tissues/organs in vehicle control (G1) and high dose (G4) group animals were subjected to histopathological examination.

No treatment related clinical signs of toxicity or mortality was observed in any of the vehicle or test item administered animals up to the high dose group of 200 mg/kg.

No treatment related changes in body weight, percent change in body weight with respect to day 1, feed consumption, functional/neurological examination and ophthalmoscopic examination were noted. No adverse treatment related changes were noted in haematology, clinical chemistry and urinalysis parameters. No toxicologically significant changes were noted infasting body weights,organ weights and its ratios. There were no statistically significant variations noted in any of the thyroid hormone assessed (T3, T4 and TSH) when compared with vehicle control group.

No treatment related gross and histopathology changes were noted in any organ and/or tissues.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

200 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

reliable without restriction

System:

other: no adverse effects observed

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

In the absence of any evidence for species specific effects or modes of action the effects observed in animals are regarded as relevant for humans.

Additional information

Oral:

Relevant NOAEL (OECD 408, gavage, rat): 200 mg/kg bw/d

 

Phosphoric acid, mixed esters with butyl alcohol and ethylene glycol was administered daily in graduated doses to 3 groups of test animals. Animals of an additional control group were handled identically as the dose groups but received sterile water, the vehicle used in this study. The 4 groups comprised 10 male and 10 female Wistar rats.

No treatment related clinical signs of toxicity or mortality was observed in any of the vehicle or test item administered animals up to the high dose group of 200 mg/kg bw/d. No treatment related changes in body weight, % change in body weight with respect to the day 1, feed consumption, functional/neurological examination and ophthalmoscopic examination, were noted. No adeverse effect was reported in hematology, clinical chemistry and urinalysis parameters. No toxicologically significant significant changes were noted in fasting body weights, organ weights and its ratios. There were no statisitcally significant variations noted in any of the thyroid hormone assessed (T3, T4 and TSH) when compared with the vehicle group. No treatment related changes in gross pathology and histopathology were reported in any organ/or tissue.

 

Based on the results of this study, the no-observed-adverse-effect-level (NOAEL) for the test item, Phosphoric acid, mixed esters with butyl alcohol and ethylene glycol, was considered to be 200 mg/kg body weight/day.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
The OECD 408 study was selected as relevant available repeated dose toxicity study due to its reliability and since it provides a sensitive NOAEL.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
In accordance with column 2 of REACH Annexes VIII and IX, the repeated dose toxicity study, as required in section 8.6.1 of Annex VIII and in section 8.6.2 of Annex IX, does not need to use the inhalation route because exposure of human via inhalation, especially in a higher extent than via oral application as performed in the animal studies, is considered unlikely taking into account the vapour pressure of the substance and the physical form (viscous liquid).

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
In accordance with column 2 of REACH Annexes VIII and IX, the repeated dose toxicity study, as required in section 8.6.1 of Annex VIII and in section 8.6.2 of Annex IX, does not need to use the inhalation route because exposure of human via inhalation, especially in a higher extent than via oral application as performed in the animal studies, is considered unlikely taking into account the vapour pressure of the substance and the physical form (viscous liquid).

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
In accordance with column 2 of REACH Annexes VIII and IX, the repeated dose toxicity study, as required in section 8.6.1 of Annex VIII and in section 8.6.2 of Annex IX, does not need to use the dermal route because
- no systemic effects or other evidence of absorption were observed in skin irritation studies in rabbits as well as in the local lymph node assay in mice
- due its irritant properties only local skin effects are expected to occur; however, since the edema/erythema were reversible these are considered to have no impact on the absorption of the test substance via skin. Due to the combination of polar (ionic) and unipolar components of the mixture it is unlikely that higher amounts than tested in a repeated oral toxicity study will be systemically available via the skin barrier.

Justification for selection of repeated dose toxicity dermal - local effects endpoint:
In accordance with column 2 of REACH Annexes VIII and IX, the repeated dose toxicity study, as required in section 8.6.1 of Annex VIII and in section 8.6.2 of Annex IX, does not need to use the dermal route because
- no systemic effects or other evidence of absorption were observed in skin irritation studies in rabbits as well as in the local lymph node assay in mice
- due its irritant properties only local skin effects are expected to occur; however, since the edema/erythema were reversible these are considered to have no impact on the absorption of the test substance via skin. Due to the combination of polar (ionic) and unipolar components of the mixture it is unlikely that higher amounts than tested in a repeated oral toxicity study will be systemically available via the skin barrier.

Justification for classification or non-classification

Due to the NOAEL of 200 mg/kg bw/day in an OECD 408 repeated dose toxicity screening test in rats, Phosphoric acid, mixed esters with butyl alcohol and ethylene glycol does not have to be classified regarding systemic and target organ toxicity after repeated exposure according to the criteria laid down in the EU Classification Labelling and Packaging Regulation (1272/2008/EC).